Ca2+-independent and Ca2+-inhibited membrane binding by synaptotagmin-like proteins Project Summary/Abstract Protein-membrane interactions underlie most of the important steps in vesicle trafficking and exocytosis, including for hormones such as insulin. Although many of the key proteins are known, a better understanding of the structural mechanisms of membrane interaction is necessary for the future design of selective inhibitors. The broad goal of this study is to provide such a mechanistic and structural understanding for synaptotagmin- like proteins (Slp), a key family of proteins whose inhibition may be pharmaceutically useful. Two representative and yet unique members of the synaptotagmin-like protein (Slp) family will be investigated: Slp-2 and Slp-4. These have been chosen as our focus because (i) the structure of the membrane-binding C2A domain from Slp-4 is known, and (ii) Slp-2 exhibits an intriguing and unusual property of Ca2+-inhibited membrane binding. Generally, Slp proteins function to bridge between secretory vesicles and the plasma membrane, stably docking secretory vesicles prior to exocytosis. Slp proteins possess an N-terminal domain that bind Rab GTPases on secretory vesicles, and one or two C-terminal C2 domains that dock with high affinity to plasma membranes via interaction with anionic lipid molecules such as phosphatidylinositol-(4,5)- bisphosphate (PIP2) and phosphatidylserine (PS). Most Slp proteins, including Slp-4, bind membranes independently of calcium ions; however, the membrane affinity of Slp-2 is uniquely inhibited by calcium. This project is based on the hypothesis that high-affinity Slp C2A?membrane interaction involves multiple binding sites for lipids and/or other ligands, one of which acts allosterically and is the basis for calcium inhibition in Slp-2. Specific Aim 1 asks, ?Which residues of the Slp-4 C2A domain are required for high-affinity membrane docking?? This question will be answered using a combination of experimental and computational approaches based on the known molecular structure of this protein domain. Molecular dynamics simulations will predict how different regions of the protein domain act together to interact with membrane lipids. Effects of site- directed mutagenesis will be tested experimentally using both quantitative liposome binding assays and cell- based model systems of secretion. Based on previous reports, it is predicted that mutants which substantially weaken membrane binding will enhance insulin secretion relative to the wild-type protein. Specific Aim 2 asks, ?Does a pocket conserved among synaptotagmin-like proteins inhibit lipid binding allosterically?? For this Aim, the structural mechanism of Slp-2 C2A inhibition by calcium will be probed using fluorescence-based lipid and calcium binding measurements and electron paramagnetic resonance. The goal is to develop a structural model for how calcium binding at one site inhibits membrane binding at a distal site. If calcium binding to the Slp-2 C2A domain induces a structural change that blocks membrane association, then the corresponding vestigial pocket in Slp-4 would be a potential target for discovering inhibitory compounds. Because Slp-4 limits insulin secretion in vivo, its inhibition represents a possible avenue for future diabetes therapy.